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A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
Published on: December 27, 2013
La neurotoxina botulínica B reconoce su receptor de proteínas con alta afinidad y especificidad
Rongsheng Jin1, Andreas Rummel, Thomas Binz
1Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA.
Nature
|December 15, 2006
Resumen
Las neurotoxinas botulínicas (BoNT) se unen a las células a través de la sinaptotagmina II y los gangliosidos. Esta visión estructural de la unión BoNT/B avanza en el desarrollo de vacunas e inhibidores contra esta neurotoxina.
Área de la Ciencia:
- La neurociencia es la neurociencia.
- Biología Estructural Biología estructural.
- La bioquímica es la bioquímica.
Sus antecedentes:
- Las neurotoxinas botulínicas (BoNT) son proteínas altamente tóxicas producidas por el Clostridium botulinum.
- Los BoNT causan botulismo al bloquear la liberación de neurotransmisores en las uniones neuromusculares.
- Los mecanismos moleculares precisos de la entrada de las células BoNT y la unión al receptor no están claros.
Objetivo del estudio:
- Para aclarar los detalles moleculares del reconocimiento del serotipo B (BoNT/B) de la neurotoxina botulínica por su receptor de proteínas.
- Para determinar la estructura de alta resolución de BoNT/B en complejo con su receptor, sinaptotagmina II.
- Comprender el papel sinérgico de la sinaptotagmina II y los gangliosidos en la unión y translocación de BoNT/B.
Principales métodos:
- Se utilizó cristalografía de rayos X para determinar la estructura de BoNT/B unido al dominio luminal de la sinaptotagmina II a una resolución de 2,15 Å.
- Se realizaron ensayos bioquímicos y estudios neuronales ex vivo utilizando mutaciones basadas en la estructura.
- Análisis de la afinidad de unión y la selectividad a través de diferentes isoformas de sinaptotagmina y condiciones de pH.
Principales resultados:
- La estructura revela cómo la sinaptotagmina II induce una hélice al unirse a una grieta en BoNT/B, adyacente al sitio de unión al gangliosido.
- La sinaptotagmina II se une a BoNT/B con afinidad nanomolar tanto en pH neutro como ácido.
- Las mutaciones basadas en la estructura confirmaron una alta especificidad y afinidad por la sinaptotagmina II, con selectividad sobre la sinaptotagmina I.
- La unión sinérgica de la sinaptotagmina II y los gangliosidos restringe la translocación de BoNT/B después de la endocitosis.
Conclusiones:
- La estructura determinada proporciona una base molecular para el reconocimiento de BoNT/B por la sinaptotagmina II.
- La comprensión de la interacción del receptor dual es crucial para la entrada de células BoNT/B.
- Estos hallazgos allanan el camino para el diseño racional de nuevas terapias, incluidas vacunas e inhibidores, contra BoNTs.
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